Summary of Work: Functional genomics elucidates structure and function of proteins encoded by human genes. We developed genetic-based approaches in yeast to understand functions of human genes that may have a role in DNA and chromosome metabolism and genome stability. The human cyclins A1 and A2 genes alter phenotype in a yeast replication mutant. We also determined a genetic background in which expressed hRad51p results in synthetic lethality, providing for molecular characterization of hRad51p and its interactions with other human proteins cloned into yeast (eg. BRCA1 and p53). The human Fen1 flap endonuclease involved in DNA replication & repair was able to complement a yeast rad27 mutant for mutagen sensitivity, genetic instability and synthetic lethal interactions with repair and replication mutants. We identified a biochemically inactive form of Fen1 with dominant genotoxic effects. The genetic impacts of Fen1 in yeast is stronger when the human and yeast PCNA binding domains are exchanged. Our study defines a system to study potentially deleterious mutations in human FEN1. In collaborations with the Kunkel lab, a yeast model for functional analysis of human Msh2/Msh6 protein was developed, based on the strong mutator effect of the co-overexpressed human proteins in yeast. Functional analysis of human p53 has been pursued using established and newly developed yeast assays. The transactivation potential of p53 genes cloned directly in yeast from tumor samples and cell lines has been assessed. We exploited variable expression of human p53 in yeast and random PCR mutagenesis of the DNA binding and tetramerization domains to develop screens for p53 alleles with novel phenotypes. p53 mutants were identified that kill yeast at modest expression levels. One of these p53 toxin mutants prevented growth of human cells. A second screen was aimed at isolating p53 variants with an increased transactivation potential compared to the wild type. Both categories of p53 mutant alleles will be useful in studies aimed at dissecting the complex p53 downstream pathways and in developing cancer therapies. - Yeast, Human Gene, Phenotype Disruption, DNA Recombination, Replication, DNA Repair